The Effect of Mutation of the hFSHR Caveolin Binding Motif on Localization Within Lipid Rafts

Unintended pregnancies are a significant global health issue. Between 2015 and 2019, an estimated 121 million unintended pregnancies occurred worldwide each year, with approximately 61% ending in abortion. As access to safe and legal abortion varies across countries, effective and accessible contraceptive options remain critically important. While hormonal oral contraceptives are among the most widely used forms of birth control, they are not suitable for everyone, highlighting the need for alternative approaches, especially given that the responsibility for contraception has historically fallen on women. Due to its role in fertility, follicle-stimulating hormone (FSH) signaling represents a potential target for reducing unintended pregnancies. FSH is produced by the anterior pituitary and acts on gonadal cells to regulate reproductive processes by stimulating follicular development in females and spermatogenesis in males. The receptor for FSH (FSHR) is a g protein-coupled receptor (GPCR) located in the plasma membrane of granulosa cells in the ovaries and Sertoli cells in the testes, where it mediates these reproductive functions. Previous work in our lab has shown that human FSHR (hFSHR) localizes to cholesterol and sphingolipid rich microdomains of the membrane called lipid rafts. Some lipid rafts contain the scaffolding protein caveolin, which helps localize GPCRs to these domains. Direct interactions between FSHR and caveolin have been observed and are thought to be mediated by a conserved caveolin-binding motif (CBM) within the fourth transmembrane domain of hFSHR. This motif contains four phenylalanine residues believed to be critical for caveolin association. To investigate the role of these residues, mutant FSHR constructs were generated in which 3-4 phenylalanines were replaced with leucine. These mutations were analyzed to determine whether disrupting the CBM alters receptor localization within the membrane. Sucrose gradient fractionation was used to separate lipid raft from non-raft membrane fractions, followed by western blotting to compare the distribution of mutant and wild-type receptors. Mutation of all four phenylalanine residues resulted in loss of FSHR from lipid raft fractions, indicating that all aromatic residues are required for receptor localization. Understanding how membrane localization influences FSHR function may help guide the development of FSH-targeted contraceptive strategies.